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Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes

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Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes This information is current as Rasheed Ahmad, Judith Sylvester, Mushtaq Ahmad and of September 24, 2021. Muhammad Zafarullah J Immunol 2009; 182:5081-5087; ; doi: 10.4049/jimmunol.0803544 http://www.jimmunol.org/content/182/8/5081 Downloaded from References This article cites 63 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/182/8/5081.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/185/1/769.full.pdf The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes1 Rasheed Ahmad,* Judith Sylvester,* Mushtaq Ahmad,† and Muhammad Zafarullah2* Aggrecanases (a dystrophin and metalloproteinase with thrombospondin motif, ADAMTSs) are principal proteases involved in cartilage extracellular matrix aggrecan degradation. The role and relative contribution of MyD88, IRAK1, and TRAF6 adaptor proteins in IL-1␤ regulation of aggrecanase-1 (ADAMTS-4) is unknown. By small interfering RNAs-mediated knockdown, we show that IL-1␤-induced up-regulation of ADAMTS-4 in chondrocytes requires MyD88, IRAK1, and TRAF6 adaptor proteins. However, partial inhibition of ADAMTS-4 induction by their knockdown suggested the involvement of additional signaling proteins. Because IL-1␤ is also known to induce reactive oxygen species (ROS) through Ras-mediated activation of NADPH oxidase, we investigated the implication of Ras in ADAMTS-4 regulation. Ras knockdown, or inhibition of ROS by antioxidants ␤ along with the ablation of MyD88, IRAK1, or TRAF6 more potently down-regulated IL-1 -induced ADAMTS-4. In addition, Downloaded from IL-1␤-induced phosphorylation of downstream effectors, I␬B kinase ␣␤,I␬B␣, and activation of transcription factor NF-␬B was significantly reduced in the MyD88-, IRAK1-, TRAF6-, or Ras-deficient cells. The combined knockdown of Ras and individual adaptor proteins strongly blocked the activation of IKK␣␤,I␬B␣, and NF-␬B. These findings suggest that Ras, ROS along with MyD88, IRAK1, or TRAF6 synergistically mediate ADAMTS-4 regulation by IL1-␤. Thus, complete ablation of ADAMTS-4 induction could be achieved by combined inhibition of Ras and individual adaptor proteins, which may be of therapeutic value in arthritis. The Journal of Immunology, 2009, 182: 5081–5087. http://www.jimmunol.org/ ntegrity of cartilage is essential for weight bearing ability, gies including neutralizing Abs and receptor antagonists are mobility, and flexibility of joints. Chondrocytes synthesize aimed at inhibiting TNF-␣ and IL-1 actions (7, 8). IL-1 binds to I and maintain cartilage extracellular matrix (ECM),3 which its receptor, TLR/IL-1R1, leading to complexation with IL-R is principally made up of type II collagen and proteoglycan accessory protein, recruitment of MyD88 intracellular adaptor aggrecates or aggrecan whose function is to resist compression. protein, interaction with IL-1R-associated kinase (IRAK), its Aggrecan monomers consist of a core protein from where chon- phosphorylation, and association with TNF receptor-associated droitin sulfate and keratan sulfate chains protrude. Interaction factor 6 (TRAF6). These events lead to activation of multiple of aggrecan hyluronon with link protein constitutes large ag- cascades including ERK, p38, JNK, AP-1, and NF-␬B pathways by guest on September 24, 2021 gregates (1). Due to their ability to inhibit ECM synthesis (2), (7, 9). ␣ proinflammatory cytokines such as IL-1 and TNF- produced Upon IL-1 exposure, aggrecan is destroyed first, followed by by inflammatory cells are considered as prime instigators of the digestion of collagen fibrils. Matrix metalloproteinase-13 cartilage ECM degradation during the pathogenesis of rheuma- (MMP-13 or collagenase-3) preferentially cleaves type-II col- toid arthritis (RA) and osteoarthritis (OA) (3, 4). Levels of IL-1 lagen while a family of aggrecanases or ADAMTSs (a dystro- are augmented in the synovial fluid and cartilage of patients phin and metalloproteinase with thrombospondin motif) is in- with arthritis (5, 6) and some of the major anti-arthritic strate- volved in physiological remodeling and pathological cleavage of aggrecan at the Glu373-Ala374 bond of interglobular domain *Department of Medicine, University of Montreal and Centre de Recherche du Centre of the core protein (10, 11). ADAMTS-4 is expressed in active Hospitalier de l’Universite´ de Montre´al, Notre-Dame Hospital, Montreal, Quebec, form in OA cartilage and aggrecan fragments are found in OA Canada; and †Cardiovascular Research Institute (CVRI), Morehouse School of Med- icine, Atlanta, GA 30310 synovial fluid (12, 13). ADAMTS-5 and not ADAMTS-4 Received for publication October 22, 2008. Accepted for publication February knockout resulted in inhibition of cartilage aggrecan destruc- 9, 2009. tion, suggesting ADAMTS-5 as the predominant aggrecanase in The costs of publication of this article were defrayed in part by the payment of page mice (14, 15). However, ex vivo RNA interference experiments charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. with the human cartilage explants revealed that both ADAMTS-4 and Ϫ5 are required for human cartilage aggrecan degradation (16). 1 This work was supported by a grant (to M.Z.) and a fellowship (to R.A.) from the Canadian Institutes of Health Research and start-up funds (to M.A.) from Morehouse Blocking aggrecanolysis in the interglobular domain by genetic School of Medicine. means resulted in abrogation of cartilage erosion (17). Although 2 Address correspondence and reprint requests to Dr. M. Zafarullah, K-5255 Mail- implication of protein kinase C in aggrecanase has been dem- loux, Hoˆpital Notre-Dame du Centre Hospitalier de l’Universite´ de Montre´al, 1560 Sherbrooke est, Montre´al, Que´bec, Canada H2L 4M1. E-mail address: onstrated (18, 19), the detailed mechanism of IL-1 signal trans- [email protected] duction leading to ADAMTS-4 gene induction and the role of 3 Abbreviations used in this paper: ECM, extracellular matrix; RA, rheumatoid adaptor proteins in particular is not known. By RNA inter- arthritis; OA, osteoarthritis; ADAMTS, A dystrophin and metalloproteinase with ference-mediated genetic knockdown, we demonstrate that thrombospondin motif; IRAK1, IL-1R-associated kinase; TRAF6, TNF receptor- associated factor 6; siRNA, small interfering RNA; ROS, reactive oxygen species; MyD88, IRAK-1, and TRAF6, as well as Ras-driven reactive IKK, I␬B kinase; MMP-13, matrix metalloproteinase-13; NDGA, nordihydroguai- oxygen species (ROS) culminating on NF-␬B transcription fac- aretic acid. tor synergistically regulate IL-1-induced ADAMTS-4 gene Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 expression. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803544 5082 AGGRECANASE-1 REGULATION BY MYD88, IRAK1, TRAF6 AND RAS Downloaded from http://www.jimmunol.org/ FIGURE 1. siRNA-mediated knockdown of MyD88, IRAK1, and TRAF6 expression results in the inhibition of IL-1␤-induced ADAMTS-4 gene expression and enzyme activity. A, Chondrocytes were transfected with either adaptor-specific siRNA or control siRNA. Cells were harvested 48 h later and protein lysates analyzed for MyD88, IRAK1, TRAF6, TRAF2, and ␤-actin expression by immunoblotting. Representative Western blots show specific inhibition of the target proteins. B, Control siRNA-transfected as well as MyD88-, IRAK1-, TRAF6-, and TRAF2-deficient chondrocytes were incubated with either IL-1␤ or TNF-␣. Supernatants and chondrocytes were harvested 24 h posttreatment, respectively, for total RNA extraction and ADAMTS-4 by guest on September 24, 2021 activity. ADAMTS-4 gene expression was examined by RT-PCR. C, ADAMTS-4 enzyme activity from the supernatants was determined by ELISA. The values are mean Ϯ SD of three separate experiments. Materials and Methods Western blotting Chondrocyte cultures and antioxidant treatments Chondrocytes were harvested and incubated for 30 min with lysis buffer The normal primary human knee articular chondrocytes (Cambrex) were (Tris 62.5 mM (pH 7.5), 1% Triton X-100, 10% glycerol). The lysates were grown to confluence as high-density passage 2 monolayer cultures in Dif- then centrifuged at 14000 rpm for 10 min and the supernatants were col- lected. Protein concentration in the lysates was measured by the Bio-Rad ferentiation Bullekit medium where they maintain their differentiated phe- ␮ notype. These cells express cartilage-specific type II collagen marker as Protein Assay kit. Protein (20 g) samples were mixed with sample load- analyzed by Western blot analysis. The chondrocytes were grown in six- ing buffer, heated for 5 min at 95°C and separated by SDS-PAGE. Cellular well plates in DMEM (Invitrogen) with 10% FCS. Chondrocytes were proteins were transferred to Immobilin-P membrane by electroblotting. The washed with PBS, kept in serum-free DMEM for 24 h, and preincubated membranes were then blocked with 5% nonfat milk in PBS for 1 h, incu- with Trolox (100 ␮M) or nordihydroguaiaretic acid (NDGA) (20 ␮M) bated with the desired primary Ab diluted either in 5% milk in PBS or 5% BSA overnight at 4°C.
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